Acanthamoeba releases lysosomal hydrolases continuously into the culture medium. this release is specific and energy dependent. The secreted hydrolases appear to be those which are trapped in the vesicular shuttle which returns membrane from the vacuolar compartment to the cell surface. Hydrolases secreted by pinocytosing cells separate into 2 groups on the basis of their secretion kinetics. One is secreted at about 15% of the cellular activity per hour and the other at about 5%. By contrast all of the hydrolases secreted by amoebae phagocytosing yeast have similar secretion kinetics. Molecular probes which have a pH dependent fluorescent intensity indicate that the vacuolar compartment of pinocytosing cells has a significantly lower pH than that of phagocytosing cells. Binding studies show that Acanthamoeba hydrolases exhibit a differential pH-dependent binding to phagolysosomal membranes. Together the results suggest that the different hydrolase secretion patterns occuring with cells pinocytosing soluble proteins and those phagocytosing yeast are related to differences in the pH of vacuoles which give rise to the shuttle vesicles.